Conventionally, studies in the fields of analytical biochemistry and clinical chemistry have been generally made on the basis of working with sample treatment solutions of milliliter amounts. With recent development of biotechnology and immunochemistry, however, the studies in these fields are made on the basis of results of treatment of sample solutions of size on the order of microliters. This is because in many instances, only microliters of solution are available, or because so many different analyses must be undertaken that a larger original sample, on the order of 1-10 milliliters must be subdivided into a large number of aliquots, so that each aliquot is only a fraction of a milliliter. Further, in some instances, the biological target molecule of interest is present in such dilution that many repeated iterations of concentration or amplification must be undertaken before enough of the target sample is obtained for meaningful qualitative or quantitative analysis. The result of the concentration is, likewise, usually only a microliter quantity of solution for treatment after the subdivision into test aliquots, as many different tests, screenings or treatments must be effected to identify or characterize the target molecule. Working on a microscale has introduced a whole variety of new and extremely complex problems, particularly in the quantitative arena as the treatment unit of the sample solution becomes smaller.
In the analysis of biological samples by high performance liquid chromatography (HPLC), high performance capillary zone electrophoresis or many other techniques, pretreatment of a samples prior to analysis is often required. In other cases, two or more enzymatic digestions must be conducted in succession to obtain the desired products. In such instances, it is necessary for the sample solution, obtained by an enzyme reaction in a reaction tube, to be filtered through an ultrafiltration membrane to remove molecules having larger molecular weights or insoluble fine particles in order to prevent clogging of the high performance liquid chromatography columns.
For example, in a typical procedure for reducing oligosaccharides from a glycoprotein for analysis by high performance anion exchange chromatography (HPAEC) or high performance liquid chromatography (HPLC) after derivatization, the following steps are usually required: (1) reduction and alkylation; (2) dialysis; (3) freeze-drying; (4) digestion with a suitable protease; (5) gel filtration to enrich glycopeptides; (6) digestion with an enzyme to release oligosaccharides; and (7) separation of peptides and oligosaccharides to minimize interference. Between each of these steps a transfer of the sample solution is required.
Typically, an instrument, such as for example a micropipet, is used to transfer the sample solution from the reaction microtube into another device for ultrafiltration. In this method, however, a certain amount of loss of the sample is inevitable, for example when in the process of transferring the sample, solution is pipetted from one test microtube or vial to another, the quantity of solution which is left behind clinging to the pipette is so large that the quantitative analysis may be completely thrown off. The loss is greater when the sample quantities are smaller. In such treatments of micro-samples in microliters as described above, the effects of such a loss of sample cannot be neglected. Thus, new systems have been developed which are pipette-less to avoid the solution loss during transfer or analysis problems.
In a second example, a protein may be labeled using radioisotopes, and then the labeled protein constituent and the isotopes should be separated. In such cases, it is conventional that, after labeling with the isotope in a reaction tube, part or all of the sample solution is transferred, by micropiper or the like, into a device for radiation measurement. Accordingly, the above-described problem of loss of the sample also arises in the process of transferring the sample solution. Furthermore, the risk of radiation contamination of instruments used in liquid transfer cannot be avoided.
As described above, in the conventional handling method of sample solutions there exist problems of loss of sample and contamination of instruments. These problems cannot be avoided when transferring the sample solution from the reaction microtube into various kinds of solution treatment devices. Furthermore, when carrying out sample handling procedures which by their nature require a plurality of steps, such as the enzyme reaction and the sample radio-isotope labeling procedures described above, the problems associated with the amount of sample loss and degree of instrument contamination get progressively worse, since these sample handling procedures require multiple transfers of the sample.
A third problem lies in proper delivery of the quantitatively required amount of reagents of inorganic, organic and biochemical natures to the target solutions in order to effect the various treatment reactions to the target solutions. Again, the pipette effect is extremely significant. It is difficult to compensate for the pipette effect because the amount of solution which is left behind clinging to the pipette varies by the nature of the solvent and solute to some extent, and often to a greater extent by the technique of the person doing the laboratory manipulation. It is also inconsistent, because even the most experienced laboratory technician can have momentary lapses or interruptions which introduce irregularities.
In systems involving micro-filter centrifugation, the problem is also heightened because the solution left behind in one vial may have a very large effect. In addition, some of the reagents must be applied to the filter media between the two vials so that the reaction or treatment occurs as the filtrate liquid is passing through the membrane, and it is important that all of the liquid be treated. In other instances, the treatment must occur in connection with the liquid after filtration, because the filter must be used to retain non-treated or previously treated biological molecules, cells or other material.
U.S. Pat. No. 4,632,761 issued to Bowers et al., discloses a centrifugal microconcentrator assembly comprising a sample reservoir (source tube) and a filtrate cup (target tube) joined together at their openings by a connector assembly which contains a filter membrane for use in concentrating macromolecules from a sample solution. The connector assembly has a first end adapted for crimp sealing to the outer periphery of the reservoir opening and a second end adapted for plug insertion into the opening of the filtrate cup. In operation, the microconcentrater is placed in a centrifuge rotor with the filtrate cup (target tube) facing down, and is centrifuged such that the sample solution is transferred from the sample reservoir (source tube) through the filter membrane and into the filtrate cup (target tube). A disadvantage with this device occurs when repetitive filtration or treatment steps are desired, since the sample solution recovered in the filtrate cup (target tube) must be transferred somehow to a new sample reservoir (target tube). As discussed above, a micropipet is typically used for this purpose and the problem of material loss of sample occurs.
In addition, microconcentrator tubes do not readily fit into centrifuge wells. Many microcentrifuge designs are so small that such longer microconcentrator tubes also interfere with covering lids or with oppositely located tubes when placed in the rotor. Or they may, under the gravitational effect of centrifuging, tilt or cant to one side and spill, or the tips of the tubes touch bottom and become cracked or crushed and leak. All of these are consequences of design for one purpose that overlooks problems raised by such design in actual practice.
Accordingly, there is a definite need in the art for a connection-type centrifugal micro solution treatment system which includes a universal connection assembly for joining together a source microtube and target microtube in interchangeable fashion to permit repeated filtrations or treatments of a sample solution back and forth between the two microtubes without a significant loss of sample, and for adapters which permit retrofit usage in commercially available centrifuges without need for complete redesign of centrifuge rotors or covers.
In another biochemical arena, proteins exhibit a wide range of biological properties, particularly therapeutic properties in ameliorating various adverse medical conditions or diseases. There has arisen an entire field of characterizing the structure of such proteins. This is done by subjecting the proteins to repeated reactions to disassemble the constituent amino acids (herein AAs). A principal method is to use proteases, which are usually natural enzymes that can sever the peptide bonds between adjacent amino acids. Some proteases are highly site-specific, and can be used to fragment a protein into specific AAs or peptide fragments for sequence analysis.
Conversely, there is an entire biochemical/biopharmacological field of creating new peptides and proteins which are then assayed for biological binding activity against target molecules that have adverse biologic activity. A typical approach is to create vast, random, hexapeptide screening libraries of at least a substantial number of the 64 million possible hexapeptide combinations of the 20 L-amino acids, determining which are active in an iterative sequence, and then characterizing the sequence of the unknown active hexapeptides. In the iterative process it is common to build the hexapeptide one or two AAs at a time in a manner that requires some be blocked and others unblocked, at different times, so that all the possible random combinations of the hexapeptides can be assembled. This is called N-terminal blocking, typically by acylating the terminal amino group of a di, tetra or hexapeptide that has been secured to microbeads. Proteases are used to unblock, as well as to sever the peptide bonds so the hexapeptide or smaller peptide fragment of interest can be identified, eg., by High-Performance Liquid Chromatography (HPLC) or High-Performance Capillary Electrophoresis (CZE). For examples of the peptide library formation see U.S. Pat. No. 4,631,211, which sets forth the Houghton (Iterex) T-Bag method, and U.S. Pat. No. 5,143,854 which sets forth the Pirrung et al. (Affymax) photolithographic method.
One of the problems in this field is that thousands, or hundreds of thousands of peptide/protein fragmentation reactions must be run, and each takes time and space. Present instrumentation is now highly automated and sufficiently precise that micro-quantities of solution can be handled. This saves space and prevents mind-numbing repetition-type mistakes, but it does not solve the numbers or meniscus problem. Accurate amounts of reagents must be applied to thousands and thousands of test tubes or vials. Doing that sequentially introduces significant time lapse between microtube 1, and microtube 1,000 or 10,000. And the reactants must be fresh.
Accordingly, there is a need in this biochemical field for micro-analytic systems that permit accurate, simultaneous delivery or placement of precise quantities of known reagents in arrays of thousands of reaction vials for introduction of target solutions for treatment or analysis.